Ingenieurwissenschaften und zugeordnete Tätigkeiten
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Due to its simplicity, speed and ability to obtain a particle number size distribution, single particle ICP-MS (spICP-MS) has emerged as an important tool for the analysis of nanoparticles (NPs). However, when NPs are suspended in a complex, unknown solution, matrix effects can occur affecting the instrument’s sensitivity. As a result, an over- or underestimation of the particle size is possible.
In this work, a proof-of-concept study of the combination of isotopic dilution analysis (IDA) and spICP-MS compensating for possible matrix effects is presented. As an example, an isotopically enriched 109Ag standard solution was added to silver NPs (Ag NP) suspensions. Different NP suspensions with mean particle diameters between 30 and 80 nm were chosen. The mixtures were analyzed using a quadrupole ICP-MS instrument. Both Ag isotopes (107Ag and 109Ag) were monitored during one experiment. The result show a good agreement with the diameters obtained using conventional spICP-MS.
In a second step, the Ag NPs were suspended in a simulated seawater matrix. Using conventional spICP-MS, a great reduction in the signal intensities and consequently in the particle sizes, was monitored. The application of the IDA-spICP-MS approach on these samples was able to obtain similar diameters compared to the samples without matrix.
We report on the development of ultra-small core-shell silver nanoparticles synthesized by an up-scaled modification of the polyol process. It is foreseen to use these thoroughly characterized particles as reference material to compare the catalytic and biological properties of functionalized silver nanoparticles. Small-angle X-ray scattering (SAXS) analysis reveal a narrow size distribution of the silver cores with a mean radius of RC = 3.0 nm and a distribution width of 0.6 nm. Dynamic light scattering (DLS) provides a hydrodynamic radius of RH = 10.0 nm and a PDI of 0.09. The particles’ surface is covered with poly(acrylic acid) (PAA) forming a shell with a thickness of 7.0 nm, which provides colloidal stability lasting for more than six months at ambient conditions. The PAA can be easily exchanged by biomolecules to modify the surface functionality. Replacements of PAA with glutathione (GSH) and bovine serum albumin (BSA) have been performed as examples. We demonstrate that the particles effectively catalyze the reduction of 4-nitrophenol to 4-aminophenol with sodium borohydride. With PAA as stabilizer, the catalytic activity of 436 ± 24 L g⁻¹ s⁻¹ is the highest reported in literature for silver nanoparticles. GSH and BSA passivate the surface substantially resulting in a catalytic activity of 77.6 ± 0.9 and 3.47 ± 0.50 L g⁻¹ s⁻¹, respectively.
The presence of nano-scaled particles in food and food-related products has drawn attention to the oral uptake of nanoparticles and their interactions with biological systems. In the present study, we used a toxicoproteomics approach to allow for the untargeted experimental identification and comparative analysis of cellular Responses in rat liver after repeated-dose treatment with silver nanoparticles, ions, and the coating matrix used for particle stabilization.
The proteomic analysis revealed treatment-related effects caused by exposure to silver in particulate and ionic form. Both silver species induced similar patterns of signaling and metabolic alterations. Silver-induced cellular alterations comprised, amongst others, proteins involved in metal homeostasis, oxidative stress response, and energy metabolism. However, we discovered that secondary nano-scaled structures were formed from ionic silver. Furthermore, also the coating matrix alone gave rise to the formation of nano-scaled particles.
The present data confirm, complement, and extend previous knowledge on silver toxicity in rodent liver by providing a comprehensive proteomic data set. The observation of secondary particle formation from nonparticle controls underlines the difficulties in separating particle-, ion-, and matrix coating-related effects in biological systems. Awareness of this issue will support proper evaluation of nanotoxicology-related data in the future.
Considerations using silver nitrate as a reference for in vitro tests with silver nanoparticles
(2016)
Most in vitro tests regarding the cellular toxicology of nanoparticulate metals compare particle to associated metal ion exposure. However. it is also a fact. that for example silver ions are reduced by sugars or transformed to silver chloride by chloride salts which are abundant components of cell culture media. These reactions are likely to either complicate or even invalidate comparisons between effects of ions and particles. Here. we present a fast and quantitative method to determine particle formation and numbers in different cell culture media with non-destructive small-angle X-ray scattering (SAXS). Silver nitrate with a concentration of25 (.Jg Ag mL -I was dissolved for up to 24 h at 37 'C in Dulbeccos Modified Eagle Medium (DMEM) with and without 10% fetal bovine serum (FBS) and a solution ofO-glucose (4.5 (.Jg mL -1). respectively. Silver nanopartides were observed in all Solutions after 5 min. The cell culture media displayed a limited particle-growth. FBS showed an effect on the polydispersity of the generated particles but after 5 min the overall particle size was nearly equal in FBS and non FBS supplemented medium. Particles in D-glucose were precipitating after 10 min. Particulate silver concentration was between 3 and 4 (.Jg mL -1 in both cell culture media (CCM). These results should be taken into account when performing silver ion-toxicity experiments in relevant media.
The utilization of silver nanoparticles in consumer related products has significantly increased over the last decade, especially due to their antimicrobial properties. Today they are used in a high variety of products ranging from food containers over children toys and textiles. Therefore, research on the toxicological potential of silver nanoparticles becomes increasingly important for a high amount of studies. Unfortunately, the results of these studies are extremely diverse and do not lead to a consistent evaluation. The central problem lies in the use of a wide range of silver nanoparticles, which show a broad size distribution. To overcome this problem we report on the synthesis and application of small silver nanoparticles with a narrow size distribution (R = 3.1 nm, σ = 0.6 nm). The poly(acrylic acid) stabilized particles are thoroughly characterized by small-angle X-ray scattering, dynamic light scattering and UV/Vis spectroscopy. The particles are highly stable and show no aggregation for more than six months. It is foreseen to use these thoroughly characterized nanoparticles as reference material to compare the catalytic and biological properties of functionalized silver nanoparticles. As a first step the particles are used in the first world-wide inter-laboratory comparison of SAXS. Furthermore, the stabilizing ligand PAA can be easily exchanged by biomolecules to modify the surface functionality. Replacements of PAA with glutathione (GSH) and bovine serum albumin (BSA) have been performed as examples. With this flexible system first applications regarding biological application in an artificial digestion procedure have been performed. Thereby the changes in size distribution and aggregation state were monitored by SAXS. Additionally these particles show a high catalytic activity of (436 ± 24) L g-1 s-1 in the reduction of 4- nitrophenol to 4-aminophenol. This activity is two orders of magnitude higher than for other silver particles in the literature.
Creating the Silver Standard: Development of a Silver Nanoparticle Reference Material using SAXS
(2017)
The utilization of silver nanoparticles in consumer related products has significantly increased over the last decade, especially due to their antimicrobial properties. Today they are used in a high variety of products, which ranges from food containers over children toys and textiles. Therefore, research on the toxicological potential of silver in a nanoscale form becomes increasingly important for a high amount of studies. Unfortunately the results of these studies are extremely diverse and do not lead to a consistent evaluation of the toxicity of silver nanoparticles. The central problem lies in the use of a wide range of silver nanoparticles, which show a broad size distribution. To overcome this problem we report on the synthesis of ultra-small silver nanoparticles and their quantitative characterization by small-angle X-ray scattering. The particles are highly stable and show no aggregation for more than six months. SAXS analysis via a Monte Carlo data evaluation procedure reveal a narrow size distribution of the silver cores with a mean volume weighted radius of 3.0 nm and a distribution width of 0.6 nm. Dynamic light scattering provides a hydrodynamic radius of 10.0 nm and a PDI of 0.09. The particles are stabilized with poly(acrylic acid) (PAA) forming a shell with a thickness of 7.0 nm. It is foreseen to use these thoroughly characterized particles as reference material to compare the catalytic and biological properties of functionalized silver nanoparticles. As a first step the particles are used in the first world-wide inter-laboratory comparison of SAXS. This study reveals that SAXS shows highly reproducible results for particles in the sub-20 nm region independently on the type of instrument used. Furthermore, the stabilizing ligand PAA can be easily exchanged by biomolecules to modify the surface functionality. Replacements of PAA with glutathione (GSH) and bovine serum albumin (BSA) have been performed as examples. With this flexible system first applications regarding biological application in an artificial digestion procedure have been performed. Thereby the changes in size distribution and aggregation state were monitored by SAXS.
Ever since increasing a reaction’s yield while shortening the reaction time is the main objective in synthesis optimization. Microwave reactors meet these demands. In literature however their usage is under discussion due to claims of the existence of non-thermal effects resulting from the microwave radiation. Especially for nano-material syntheses it is of crucial importance to be aware of influences on the reaction pathway. Therefore, we compare ultra-small silver nanoparticles with mean radii of 3 nm, synthesized via conventional and microwave heating. We employed a versatile one-pot polyol synthesis of poly(acrylic acid) (PAA) stabilized silver nanoparticles, which display superior catalytic properties. No microwave specific effects in terms of particle size distribution characteristics, as derived by small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS), are revealed. Due to the microwave reactor’s characteristics of a closed system, syntheses can be carried out at temperatures beyond the solvent’s boiling point. Particle formation was accelerated by a factor of 30 by increasing the reaction temperature from 200 °C to 250 °C. The particle growth process follows a cluster coalescence mechanism. A post-synthetic incubation step at 250 °C induces a further growth of the particles while the size distribution broadens. Thus, utilization of microwave reactors enables an enormous decrease of the reaction time as well as the opportunity of tuning the particles’ size. Possibly, decomposition of the stabilizing ligand at elevated temperatures results in reduced yields. A temperature of 250 °C and a corresponding reaction time of 30 s represent a compromise between short reaction times and high yields.
The elucidation of mechanisms underlying the cellular uptake of nanoparticles (NPs) is an important topic in nanotoxicological research. Most studies dealing with silver NP uptake provide only qualitative data about internalization efficiency and do not consider NP-specific dosimetry. Therefore, we performed a comprehensive comparison of the cellular uptake of differently coated silver NPs of comparable size in different human intestinal Caco-2 cell-derived models to cover also the influence of the intestinal mucus barrier and uptake-specialized M-cells. We used a combination of the Transwell system, transmission electron microscopy, atomic absorption spectroscopy, and ion beam microscopy techniques. The computational in vitro sedimentation, diffusion, and dosimetry (ISDD) model was used to determine the effective dose of the particles in vitro based on their individual physicochemical characteristics. Data indicate that silver NPs with a similar size and shape show coating-dependent differences in their uptake into Caco-2 cells. The internalization of silver NPs was enhanced in uptake-specialized M-cells while the mucus did not provide a substantial barrier for NP internalization. ISDD modeling revealed a fivefold underestimation of dose–response relationships of NPs in in vitro assays. In summary, the present study provides dosimetry-adjusted quantitative data about the influence of NP coating materials in cellular uptake into human intestinal cells. Underestimation of particle effects in vitro might be prevented by using dosimetry models and by considering cell models with greater proximity to the in vivo situation, such as the M-cell model.
Fate of fluorescence labels - Their adsorption and desorption kinetics to silver nanoparticles
(2018)
Silver nanoparticles are among the most widely used and produced nanoparticles. Because of their frequent application in consumer products, the assessment of their toxicological potential has seen a renewed importance. A Major difficulty is the traceability of nanoparticles in in vitro and in vivo experiments. Even if the particles are labeled, for example, by a fluorescent marker, the dynamic exchange of ligands often prohibits their spatial localization. Our study provides an insight into the adsorption and desorption kinetics of two different fluorescent labels on silver nanoparticles with a core radius of 3 nm by dynamic light scattering, small-angle X-ray scattering, and fluorescence spectroscopy. We used BSA-FITC and tyrosine as examples for common fluorescent ligands. It is shown that the adsorption of BSA-FITC takes at least 3 days, whereas tyrosine adsorbs immediately. The quantitative amount of stabilizer on the particle surface was determined by fluorescence spectroscopy and revealed that the particles are stabilized by a monolayer of BSA-FITC (corresponding to 20 ± 9 molecules), whereas tyrosine forms a multilayered structure consisting of 15900 ± 200 molecules. Desorption experiments show that the BSA-FITC-stabilized particles are ideally suited for application in in vitro and in vivo experiments because the ligand desorption takes several days. Depending on the BSA concentration in the particles surroundings, the rate constant is k = 0.2 per day or lower when applying first order kinetics, that is, 50% of the BSAFITC molecules are released from the particle’s surface within 3.4 days. For illustration, we provide a first application of the fluorescence-labeled particles in an uptake study with two different commonly used cell lines, the human liver cell model HepG2 and the human intestinal cell model of differentiated Caco-2 cells.
During the last years, there has been a rapid rise in the use of nanomaterials in consumer products. Especially silver nanoparticles are frequently used because of their well-known optical and antimicrobial properties. However, the toxicological studies focusing on silver nanoparticles are controversial, either claiming or denying a specific nano-efffect. To contribute to localizing nanoparticles in toxicological studies and to investigate the interaction of particles with cells, a fluorescent marker is often used to monitor their transport and possible degradation. A major problem, in this context is the issue of binding stability of a fluorescent marker which is attached to the particle.
In order to overcome this problem we provide an investigation of the binding properties of fluorescence-labeled BSA to small silver nanoparticles. Therefore, we synthesized small silver nanoparticles which are stabilized by poly(acrylic acid). The particles are available as reference candidate material and were thoroughly characterized in an earlier study. The ligand was exchanged by fluorescence marked albumin (BSA-FITC). The adsorption of the ligands was monitored by dynamic light scattering (DLS). To verify that the observed effects on the hydrodynamic radius originate from the successful ligand exchange and not from agglomeration or aggregation we used small angle X-ray scattering (SAXS). The fluorescent particles were characterized by UV/Vis and fluorescence spectroscopy. Afterwards, desorption of the ligand BSA-FITC was monitored by fluorescence spectroscopy and the uptake of particles in different in vitro models was studied.
The particles are spherical and show no sign of aggregation after successful ligand exchange. The fluorescence intensity is quenched significantly by the presence of the silver cores as expected, but the remaining fluorescence intensity was high enough to use these particles in biological investigations. Half-life of fluorescence labeling on the particle was 21 d in a highly concentrated solution of non-labeled BSA. Thus, a very high dilution and long incubation times are needed to remove BSA-FITC from the particles. Finally, the fluorescence-labeled silver nanoparticles were used for uptake studies in human liver and intestinal cells, showing a high uptake for HepG2 liver cells and almost no uptake in differentiated intestinal Caco-2 cells. In conclusion, we showed production of fluorescence-marked silver nanoparticles. The fluorescence marker is strongly adsorbed to the silver surface which is crucial for future investigations in biological matrices. This is necessary for a successful investigation of the toxicological potential of silver nanoparticles.